Methods for using anti-MUC18 antibodies

ABSTRACT

The present invention relates generally to the generation and characterization of anti-MUC18 monoclonal antibodies. The invention further relates to the use of such anti-MUC18 antibodies in the diagnosis and treatment of disorders associated with increased activity of MUC18, in particular, tumors, such as melanomas.

RELATED APPLICATION

[0001] This application claims priority from U.S. provisionalapplication No. 60/346,414, filed on Dec. 28, 2001 and entitled “METHODSFOR USING ANTI-MUC18 ANTIBODIES”.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] Embodiments of the present invention concern antibodies bindingMUC18 antigen as well as methods and means for making and using suchantibodies.

[0004] 2. Description of the Related Art

[0005] MUC18 is a cell-surface glycoprotein originally identified as amelanoma antigen, melanoma cell adhesion molecule (MCAM), whoseexpression is associated with tumor progression and the development ofmetastatic potential. MUC18 is a 113 kDA cell surface integral membraneglycoprotein composed of a signal peptide, five immunoglobulin-likedomains, a transmembrane region, and a short cytoplasmic tail (Lehmannet al., Proc Natl Acad Sci USA, 86(24):9891-5 (1989)).

[0006] MUC18 is a member of the immunoglobulin superfamily and hassignificant sequence homology to a number of cell adhesion molecules ofthe Ig superfamily (Lehmann et al., Proc. Natl. Acad. Sci. USA,86:9891-9895 (1989)), including BEN (Pourquie et al., Proc. Natl. Acad.Sci. USA, 89:5261-5265 (1992)), neural-cell adhesion molecule (N-CAM)(Owens et al., Proc. Natl., Acad. Sci. USA, 84:294-298 (1987)),myelin-associated glycoprotein (MAG) (Lai et al., Proc. Natl. Acad. Sci.USA, 84:4337-4341 (1987)), deleted in colorectal cancer (DCC) (Hedricket al., Genes Devel., 8(10):1174-83 (1994)), and gicerin (Taira et al.,Neuron, 12: 861-872 (1994)). The expression of MUC18 has been detectedin relatively limited spectrum of normal human tissues and in a varietyof malignant neoplasms. In normal adult tissues, MUC 18 is expressed onendothelial cells, smooth muscle cells (Shih et al., Lab. Invest.,75:377-388 (1996);Sers et al., Cancer Res., 54(21):5689-94 (1994)), asubpopulation of activated T lymphocytes (Pickl et al., J. Immunol.,158:2107-2115 (1997)) and intermediate trophoblasts (Shih et al., Lab.Invest., 75:377-388 (1996)). MUC18 is also expressed on a variety ofmalignant neoplasms including smooth muscle neoplasms (Leiomyomas andleiomyosarcomas), tumors of vascular origin (angiosarcomas and Kaposi'ssarcomas), placental site trophoblastic tumors, choriocarcinomas andmelanomas (Shih et al., Clinical Cancer Res., 2:569-575 (1996); Holzmannet al., Int. J. Cancer, 39:466-471 (1987)). The expression of MUC18correlates directly with the metastatic potential of human melanomacells (Bar-Eli, M., Cancer Metastasis, 18(3):377-85 (1999)).

[0007] A number of studies have identified MUC18 as a marker of tumorprogression and metastasis in melanomas. The expression of MUC18 isabsent in normal melanocytes and benign nevi but prominent on manyprimary melanomas and in most metastatic lesions (Lehmann et al., Proc.Natl. Acad. Sci. USA, 86:9891-9895 (1989); Lehmann et al., Cancer Res.,47:841-845 (1987); Shih et al., Cancer Res., 54:2514-2520 (1994)).Importantly, MUC18 expression correlates well with tumor verticalthickness and metastasis formation, and greater than 80% of metastaticlesions express MUC18 (Lehmann et al., Proc. Natl. Acad. Sci. USA,86:9891-9895 (1989); Xie et al., Cancer Res., 57:2295-2303 (1997); Serset al., Proc. Natl. Acad. Sci. USA, 90:8514-8518 (1993); Lehmann et al.,Cancer Res., 47:841-845 (1987); Shih et al., Cancer Res., 54:2514-2520(1994). A diagram depicting the expression of MUC18 with respect toother known molecular lesions in human melanoma is presented in FIG. 1.

[0008] The expression of the transcription factors ATF-1 and CREB isupregulated in metastatic melanoma cells. However, how overexpression ofATF-1/CREB contributes to the acquisition of the metastasis is unclear.CREB/ATF-1 may play an essential role in invasion by regulating theCRE-dependent expression of the adhesion molecule MUC18 andmetalloproteinase MMP-2 (Jean et al., Mol. Cell Biochem., 212(1-2):19-28(2000)) which belongs to the MMP family known to contribute to cancersand to have a role in tumor invasion, angiogenesis, and metastasis.Tumor cells are believed to utilize the matrix degrading capability ofMMPs to spread to distant sites, and once the tumor cells havemetastasized, MMPs are thought to promote the growth of these tumorcells. The role of MUC18 in melanoma tumor progression is not completelyunderstood, but may include a role in one or more steps in themetastatic process possibly by affecting MMP-2 activation or cellmigration.

[0009] The analysis of human melanoma cell lines showed a positivecorrelation of MUC 18 expression with the ability of cells to producemetastases in nude mice (Johnson et al., Cancer Metastasis Rev.,18:345-357 (1999)). The generation of tumorigenic variants from anon-tumorigenic melanoma cell line was reported to be accompanied byinduction of MUC18 expression (Luca et al., Melanoma Res., 3:35-41(1993)). Expression of MUC18 on MUC18-negative human melanoma cell linesincreased their tumorigenicity and enhanced their metastatic capabilityin experimental tumor models (Xie et al., Cancer Res., 57:2295-2303(1997); Bani et al., Cancer Res., 56:3075-3086 (1996)). Finally,inhibition of MUC18 expression in metastases using genetic suppressorelements of MUC18 cDNA led to a decrease of the tumorigenic phenotype innude mice (Styamoorthy et al., Oncogene, 20:4676 (2001)).

[0010] Although the function of MUC18 is not fully understood, severalstudies have demonstrated a role for this protein in mediating cell-celland cell-matrix interactions by binding to an unidentified ligand (Shihet al., Cancer Res., 57:3835-3840 (1997); Johnson et al., Int. J.cancer, 73:769-774 (1997)). The expression of cell adhesion moleculeswhich mediate cell-to-cell or cell-to-matrix interactions is a tumorcell property that is essential for metastases. Accordingly,MUC18-transfected melanoma cells showed increased homotypic adhesion,increased attachment to human endothelial cells, and increased invasionthrough Matrigel-coated filters suggesting a role in tumor invasion andtrans-endothelial migration (Xie et al., Cancer Res., 57:2295-2303(1997)). Importantly, anti-MUC 18 antibodies were able to inhibit thesefunctions in the MUC 18-transfected cells (Xie et al., Cancer Res.,57:2295-2303 (1997)). Accordingly, there is a great need for anti-MUC 18antibodies that are able to inhibit the biological function of MUC18,most importantly cell proliferation and growth which may be essential totumor progression and metastasis. Such antibodies would likely interferewith the inherent ability of MUC18 to mediate cell-cell and cell-matrixinteractions. The inhibition of such activity may be possible with amonoclonal antibody targeted to MUC18. The ability to affect theprogression of tumor cells expressing MUC18 on the cell surface mayprove to be a treatment for patients with tumors or of use forprevention of metastatic disease in patients with such tumors.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the development of monoclonalantibodies that were found to bind MUC18 and affect MUC18 function. Thisapplication describes human anti-MUC18 antibodies and anti-MUC18antibody preparations with desirable properties from a therapeuticperspective, including strong binding affinity for MUC18, the ability toinhibit metastasis in vivo and the ability to promote cell survival.

[0012] One embodiment of the invention is a method of inhibiting cellproliferation or tumor metastasis associated with the expression ofMUC18 tumor antigen. The method includes the steps of providing amonoclonal antibody comprising a heavy chain amino acid, wherein theantibody has an amino acid sequence selected from the group consistingof SEQ ID NOs: 1, 5 9, 13, 17, 21, 25, 29, 33 and 37, and wherein saidmonoclonal antibody binds MUC18; contacting cells expressing MUC18 withan effective amount of the antibody; and incubating said cells and theantibody, wherein said incubation results in inhibited proliferation ofthe cells.

[0013] Another embodiment of the invention is a method of inhibitingcell proliferation or tumor metastasis associated with the expression ofMUC18 tumor antigen which includes: providing a monoclonal antibodycomprising a light chain amino acid, wherein the antibody has an aminoacid sequence selected from the group consisting of: SEQ ID NOs: 2, 6,10, 14, 18, 22, 26, 30, 34 and 38, and wherein said monoclonal antibodybinds MUC18; contacting cells expressing MUC18 with an effective amountof the antibody; and incubating the cells and the antibody, wherein theincubation results in inhibited proliferation of the cells.

[0014] In one aspect, the invention provides an anti-human MUC18monoclonal antibody which binds to and neutralizes a biological activityof at least human MUC18 or stimulates the internalization anddown-regulation of the protein. The antibody can significantly reduce oreliminate a biological activity of the human MUC18 in question.

[0015] The biological activity of the subject human MUC18 may be cellproliferation. Further, the biological activity may include angiogenesisand cell proliferation important for primary tumor growth andmetastasis, cell invasion and/or migration, and activation ofmetalloproteinase MMP-2. Even further, the biological activity mayinclude growth and metastasis of tumor cells in patients with tumors,for example, melanoma.

[0016] Also provided is an isolated nucleic acid molecule encoding anyof the antibodies described herein, a vector comprising the isolatednucleic acid molecule, a host cell transformed with the nucleic acidmolecule, and a method of producing the antibody comprising culturingthe host cell under conditions wherein the nucleic acid molecule isexpressed to produce the antibody and optionally recovering the antibodyfrom the host cell. The antibody may be of the IgG class. The isolatednucleic acid molecule preferably comprises a nucleotide sequenceencoding a heavy chain variable domain of a monoclonal antibody, whereinsaid nucleotide sequence is selected from the group consisting of thenucleotide sequence of the heavy chain variable domain of c3.19.1 (SEQID NO: 3), c6.11.3 (SEQ ID NO: 7), C3.10 (SEQ ID NO: 11), C3.22 (SEQ IDNO: 15), C3.27 (SEQ ID NO: 19), C3.45 (SEQ ID NO: 23), C3.65 (SEQ ID NO:27), C6.1 (SEQ ID NO: 31), C6.9 (SEQ ID NO: 35) or C6.2 (SEQ ID NO: 39),or a nucleotide sequence encoding a light chain variable domain of amonoclonal antibody, wherein said nucleotide sequence is selected fromthe group consisting of the nucleotide sequence of the light chainvariable domain of 3.19.1 (SEQ ID NO: 4), 6.11.3 (SEQ ID NO: 8), C3.10(SEQ ID NO: 12), C3.22 (SEQ ID NO: 16), C3.27 (SEQ ID NO: 20), C3.45(SEQ ID NO: 24), C3.65 (SEQ ID NO: 28), C6.1 (SEQ ID NO: 32), C6.9 (SEQID NO: 36), or C6.2 (SEQ ID NO: 40).

[0017] In a different aspect, the invention provides a method for thetreatment of a disease or condition associated with the expression ofMUC18 in a patient, comprising administering to the patient an effectiveamount of an anti-MUC18 antibody. The patient is a mammalian patient,preferably a human patient. The disease is a tumor, such as melanoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a diagram depicting the expression pattern of MUC18 andother known oncogenes and growth factors involved in melanoma tumorprogression.

[0019]FIG. 2 shows immunoblot analysis with anti-MUC18 antibodies anddemonstrates a positive correlation between MUC18 expression with themetastatic capacity of human melanoma cells. The expression of MUC18 inhuman metastatic melanoma cell lines (A375SM, TXM-13, and WM2664),nonmetastatic cell line SB-2, and normal mouse endothelial (NMEs) cellsare shown.

[0020]FIGS. 3A and 3B are line graphs illustrating that neither theA375-SM (FIG. 3A) nor the WM-2664 cells (FIG. 3B) demonstrated afluorescent shift when incubated in the presence of the control IgG2 Ab(bold line). However, when incubated in the presence of anti-MUC18(dotted line), a strong shift in fluorescence intensity indicative ofcell surface expression of the antigen was observed.

[0021]FIG. 4 is a line graph illustrating that treatment with anti-MUC18antibody c3.19.1, prolongs the survival of WM2664 mice bearingmetastatic melanoma tumors.

[0022]FIG. 5 shows the amino acid sequence of the variable region of theheavy (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) and the nucleotidesequence encoding the variable region of the heavy (SEQ ID NO: 3) andlight (SEQ ID NO: 4) chain of anti-MUC18 antibody, c3.19.1.

[0023]FIG. 6 shows the amino acid sequence of the variable region of theheavy (SEQ ID NO: 5) and light chain (SEQ ID NO: 6) and the nucleotidesequence encoding the variable region of the heavy (SEQ ID NO: 7) andlight (SEQ ID NO: 8) chain of anti-MUC18 antibody, c6.11.3.

[0024]FIG. 7 shows the amino acid sequence of the variable region of theheavy (SEQ ID NO: 9) and light chain (SEQ ID NO: 10) and the nucleotidesequence encoding the variable region of the heavy (SEQ ID NO: 11) andlight (SEQ ID NO: 12) chain of anti-MUC18 antibody, c3.10.

[0025]FIG. 8 shows the amino acid sequence of the variable region of theheavy (SEQ ID NO: 13) and light chain (SEQ ID NO: 14) and the nucleotidesequence encoding the variable region of the heavy (SEQ ID NO: 15) andlight (SEQ ID NO: 16) chain of anti-MUC18 antibody, c3.22.

[0026]FIG. 9 shows the amino acid sequence of the variable region of theheavy (SEQ ID NO: 17) and light chain (SEQ ID NO: 18) and the nucleotidesequence encoding the variable region of the heavy (SEQ ID NO: 19) andlight (SEQ ID NO: 20) chain of anti-MUC18 antibody, c3.27.

[0027]FIG. 10 shows the amino acid sequence of the variable region ofthe heavy (SEQ ID NO: 21) and light chain (SEQ ID NO: 22) and thenucleotide sequence encoding the variable region of the heavy (SEQ IDNO: 23) and light (SEQ ID NO: 24) chain of anti-MUC18 antibody, c3.45.

[0028]FIG. 11 shows the amino acid sequence of the variable region ofthe heavy (SEQ ID NO: 25) and light chain (SEQ ID NO: 26) and thenucleotide sequence encoding the variable region of the heavy (SEQ IDNO: 27) and light (SEQ ID NO: 28) chain of anti-MUC18 antibody, c3.65.

[0029]FIG. 12 shows the amino acid sequence of the variable region ofthe heavy (SEQ ID NO: 29) and light chain (SEQ ID NO: 30) and thenucleotide sequence encoding the variable region of the heavy (SEQ IDNO: 31) and light (SEQ ID NO: 32) chain of anti-MUC18 antibody, c6.1.

[0030]FIG. 13 shows the amino acid sequence of the variable region ofthe heavy (SEQ ID NO: 33) and light chain (SEQ ID NO: 34) and thenucleotide sequence encoding the variable region of the heavy (SEQ IDNO: 35) and light (SEQ ID NO: 36) chain of anti-MUC18 antibody, c6.9(also independently cloned as c6.12).

[0031]FIG. 14 shows the amino acid sequence of the variable region ofthe heavy (SEQ ID NO: 37) and light chain (SEQ ID NO: 38) and thenucleotide sequence encoding the variable region of the heavy (SEQ IDNO: 39) and light (SEQ ID NO: 40) chain of anti-MUC18 antibody, c6.2.

[0032]FIG. 15 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c3.10(SEQ ID NO: 9), and the amino acid sequence encoding the V4-59 region(SEQ ID NO: 41) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 42) is shown below the alignment.

[0033]FIG. 16 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c3.10(SEQ ID NO: 10), and the amino acid sequence encoding the O2 region (SEQID NO: 43) of the germline V_(k) gene used. The consensus sequence (SEQID NO: 44) is shown below the alignment.

[0034]FIG. 17 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c3.22(SEQ ID NO: 13), and the amino acid sequence encoding the V4-31 region(SEQ ID NO: 45) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 46) is shown below the alignment.

[0035]FIG. 18 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c3.22(SEQ ID NO: 14), and the amino acid sequence encoding the A30 region(SEQ ID NO: 47) of the germline V_(k) gene used. The consensus sequence(SEQ ID NO: 48) is shown below the alignment.

[0036]FIG. 19 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c3.27(SEQ ID NO: 17), and the amino acid sequence encoding the V4-59 region(SEQ ID NO: 49) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 50) is shown below the alignment.

[0037]FIG. 20 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c3.27(SEQ ID NO: 18), and the amino acid sequence encoding the A30 region(SEQ ID NO: 51) of the germline V_(k) gene used. The consensus sequence(SEQ ID NO: 52) is shown below the alignment.

[0038]FIG. 21 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c3.45(SEQ ID NO: 21), and the amino acid sequence encoding the V1-18 region(SEQ ID NO: 53) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 54) is shown below the alignment.

[0039]FIG. 22 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c3.45(SEQ ID NO: 22), and the amino acid sequence encoding the B3 region (SEQID NO: 55) of the germline V_(k) gene used. The consensus sequence (SEQID NO: 56) is shown below the alignment.

[0040]FIG. 23 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c3.65(SEQ ID NO: 25), and the amino acid sequence encoding the 4-31 region(SEQ ID NO: 57) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 58) is shown below the alignment.

[0041]FIG. 24 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c3.65(SEQ ID NO: 26), and the amino acid sequence encoding the O8 region (SEQID NO: 59) of the germline V_(k) gene used. The consensus sequence (SEQID NO: 60) is shown below the alignment.

[0042]FIG. 25 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c6.1 (SEQID NO: 29), and the amino acid sequence encoding the V3-30 region (SEQID NO: 61) of the germline V_(H) gene used. The consensus sequence (SEQID NO: 62) is shown below the alignment.

[0043]FIG. 26 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC1 8 antibody, c6.1(SEQ ID NO: 30), and the amino acid sequence encoding the A20 region(SEQ ID NO: 63) of the germline V_(k) gene used. The consensus sequence(SEQ ID NO: 64) is shown below the alignment.

[0044]FIG. 27 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c6.12,and the amino acid sequence encoding the V4-31 region (SEQ ID NO: 65) ofthe germline V_(H) gene used. The consensus sequence (SEQ ID NO: 66) isshown below the alignment.

[0045]FIG. 28 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c6.12,and the amino acid sequence encoding the L2 region (SEQ ID NO: 67) ofthe germline V_(k) gene used. The consensus sequence (SEQ ID NO: 68) isshown below the alignment.

[0046]FIG. 29 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c6.2 (SEQID NO: 37), and the amino acid sequence encoding the V4-59 region (SEQID NO: 69) of the germline V_(H) gene used. The consensus sequence (SEQID NO: 70) is shown below the alignment.

[0047]FIG. 30 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c6.2 (SEQID NO: 38), and the amino acid sequence encoding the A19 region (SEQ IDNO: 71) of the germline V_(k) gene used. The consensus sequence (SEQ IDNO: 72) is shown below the alignment.

[0048]FIG. 31 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c6.9 (SEQID NO: 33), and the amino acid sequence encoding the V4-31 region (SEQID NO: 73) of the germline V_(H) gene used. The consensus sequence (SEQID NO: 74) is shown below the alignment.

[0049]FIG. 32 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC1 8 antibody, c6.9(SEQ ID NO: 34), and the amino acid sequence encoding the L2 region (SEQID NO: 75) of the germline V_(k) gene used. The consensus sequence (SEQID NO: 76) is shown below the alignment.

[0050]FIG. 33 represents an alignment between the amino acid sequence ofthe variable region of the heavy chain of anti-MUC18 antibody, c6.11.3(SEQ ID NO: 5), and the amino acid sequence encoding the V4-31 region(SEQ ID NO: 77) of the germline V_(H) gene used. The consensus sequence(SEQ ID NO: 78) is shown below the alignment.

[0051]FIG. 34 represents an alignment between the amino acid sequence ofthe variable region of the light chain of anti-MUC18 antibody, c6.11.3(SEQ ID NO: 6), and the amino acid sequence encoding the L2 region (SEQID NO: 79) of the germline V_(k) gene used. The consensus sequence (SEQID NO: 80) is shown below the alignment.

[0052]FIG. 35 represents a summary of the sequences comprising the V, D,J and resulting N recombination regions of the MUC18 antibody clonesidentified in the present invention.

DETAILED DESCRIPTION

[0053] A. Definitions

[0054] Unless defined otherwise, technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. See, e.g. Singleton etal, Dictionary of Microbiology and Molecular Biology 2^(nd) ed., J.Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning,A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor,N.Y. 1989). For purposes of the present invention, the following termsare defined below.

[0055] As used herein, the term “MUC18” refers to the cell surfacepolypeptide that is a member of the immunoglobulin superfamily withsequence similarity to a number of cell adhesion molecules. MUC18 isalso known in the art as “MCAM”, “Mel-CAM”, or “CD146”. For purposes ofthis invention, from here on, “MUC18” is used to represent “MCAM”,“Mel-CAM”, and “CD146”.

[0056] The term “c3.19.1” as used herein refers to a fully human IgG₂monoclonal antibody directed against the MUC18 antigen. The antibody wasgenerated using XenoMouse® technology (Abgenix, Inc. Fremont, Calif.)and consists of human gamma 2 heavy and kappa light chains with amolecular weight of approximately 150 kDa. C3.19.1 is also hereinreferred to as ABX-MA1 and binds specifically to human MUC18 with highaffinity (Kd=6×10⁻¹⁰ M).

[0057] The terms “biological activity” and “biologically active” withregard to MUC18 refer to the ability of a molecule to specificallyaffect tumor progression. Preferred biological activities include theability to induce growth and metastasis of tumor cells. The effect ofMUC 18 on metastasis of tumor cells may include the ability to induceMMP-2 activation and/or cell migration. A further preferred biologicalactivity is the ability to induce animal death due to tumor burden.

[0058] The terms “biological activity” and “biologically active” withregard to anti-MUC18 antibodies refer to the ability of a molecule toinhibit the growth and metastasis of tumor cells often associated withMUC18 expression. Further, another metahcnism of action or activity foranti-MUC18 antibodies include the ability to stimulate MUC18internalization and a consequent loss of cell surface expression.Specifically, the tumor cells include tumor cells in patients withtumors.

[0059] “Polymerase chain reaction” or “PCR” refers to a procedure ortechnique in which minute amounts of a specific piece of nucleic acid,RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195issued Jul. 28, 1987. Generally, sequence information from the ends ofthe region of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol. 51:263 (1987); Erlich, ed., PCR Technology (Stockton Pres,NY, 1989). As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

[0060] “Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

[0061] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial cancer or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

[0062] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0063] “Native antibodies and immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (VH) followed by a number of constantdomains. Each light chain has a variable domain at one end (VL) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains (Chothiaet al. J. Mol. Biol. 186:651 (1985; Novotny and Haber, Proc. Natl. Acad.Sci. U.S.A. 82:4592 (1985); Chothia et al., Nature 342:877-883 (1989)).

[0064] The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity.

[0065] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called κ and λ, based on the amino acid sequences of their constantdomains.

[0066] Depending on the amino acid sequence of the constant domain oftheir heavy chains, intact antibodies can be assigned to different“classes”. There are five major classes of intact antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

[0067] The term “antibody” includes all classes and subclasses of intactimmunoglobulins. The term “antibody” also covers antibody fragments. Theterm “antibody” specifically covers monoclonal antibodies, includingantibody fragment clones.

[0068] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto polyclonal antibody preparations which include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody id directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al, Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

[0069] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and terminal or internal amino acid sequence by use ofa spinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

[0070] By “neutralizing antibody” is meant an antibody molecule which isable to eliminate or significantly reduce an effector function of atarget antigen to which is binds. Accordingly, a “neutralizing”anti-MUC18 antibody is capable of eliminating or significantly reducingan effector function which may include MUC18 dependent regulation ofcell adhesion, migration or MMP activation. The antibody can affect thefuntion of MUC18 by stimulating the internalization and degradation ofthe molecule thus effectively removing cell surface expression of theantigen.

[0071] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions both in the light-chain andheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat et al(1991). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

[0072] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0073] The term “hypervariable region” when used herein refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises amino acidresidues from a “complementarity determining region” or “CDR” (e.g.residues 24-34 (L1), 50-62 (L2), and 89-97 (L3) in the light chainvariable domain and 31-55 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain; Kabat et al., Sequences of Proteins ofImmunological Interest, 5^(th) Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 ((H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol.Biol 196:901-917 (1987)). “Framework Region” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined.

[0074] The term “complementarity determining regions” or “CDRs” whenused herein refers to parts of immunological receptors that make contactwith a specific ligand and determine its specificity. The CDRs ofimmunological receptors are the most variable part of the receptorprotein, giving receptors their diversity, and are carried on six loopsat the distal end of the receptor's variable domains, three loops comingfrom each of the two variable domains of the receptor.

[0075] The term “epitope” is used to refer to binding sites for(monoclonal or polyclonal) antibodies on protein antigens.

[0076] The term amino acid or amino acid residue, as used herein ,refers to naturally occurring L amino acids or to D amino acids asdescribed further below with respect to variants. The commonly used on-and three-letter abbreviations for amino acids are used herein (BruceAlberts et al., Molecular Biology of the Cell, Garland Publishing, Inc.,New York (3d ed. 1994)).

[0077] The term “disease state” refers to a physiological state of acell or of a whole mammal in which an interruption, cessation, ordisorder of cellular or body functions, systems, or organs has occurred.

[0078] The term “treat” or “treatment” refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

[0079] A “disorder” is any condition that would benefit from treatmentas described below. This includes chronic and acute disorders or diseaseincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include benign and malignant tumors, leukemias andlymphoid malignancies, in particular prostate, renal, ovarian, stomach,endometrial, salivary gland, kidney, colon, thyroid, pancreatic,prostate or bladder cancer, and malignant tumors, such as cervicalcarcinomas and cervical intraepithelial squamous and glandularneoplasia, renal cell carcinoma (RCC), esophageal tumors, andcarcinoma-derived cell lines. A preferred disorder to be treated inaccordance with the present invention is renal and prostate cancer. Aneven further preferred disorder to be treated is melanoma

[0080] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

[0081] “Lipofection” refers to a practical nonviral method forintroduction of genetic information into target tissues. Nonviralmethods include chemical or physical methods. Lipofection uses anelectrostatically bonded complex of positively charged lipids andnegatively charged DNA as a vector which fuses with the cell membraneand delivers DNA into the cytoplasm. Lipofection differs from viralmethods in that the efficiency of transfer of genetic information bylipofection is lower than by viral vectors and that the expression ofthe gene is transient. Alternatively, the complex of lipid and DNA ismore stable and easier to handle when compared to viral vectors.

[0082] B. Methods for Carrying Out an Embodiment of the Invention

[0083] 1. Generation of Anti-MUC18 Antibodies

[0084] A description follows as to exemplary techniques for theproduction of the antibodies used in accordance with the presentinvention.

[0085] (a) Monoclonal Antibodies

[0086] Monoclonal Antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0087] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the protein used forimmunization. Alternatively, cells expressing the antigen of interestmay be used for immunization. Further alternatively, lymphocytes may beimmunized in vitro. Animals are immunized against the immunogenicconjugates or derivatives by combing 1 mg or 1 μg of conjugate (forrabbits or mice, respectively) with 3 volumes of Freud's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with ⅕ to {fraction (1/0)} theoriginal amount of conjugate in Freud's complete adjuvant bysubcutaneous injection at multiple sites. 7 to 14 days later the animalsare bled and the serum is assayed for anti-MUC18 antibody titer.Antibodies are boosted until the titer plateaus. Preferably, the animalis boosted with the conjugate of the same MUC18 antigen, but conjugatedto a different protein and/or through a different cross-linking agent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are used to enhance theimmune response.

[0088] Lymphocytes or more preferably, lymphocytes enriched for B cellsisolated from such immunized animals are then fused with myeloma cellsby an electrocell fusion process or by using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-109, [AcademicPress, 1996]).

[0089] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0090] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred mycloma cell lines are murine myelomalines, such as those derived from MOP-21 and MC.-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol. 133: 3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63, Marcel Dekker, Inc., New York, [1987]).

[0091] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

[0092] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal. Biochem.107: 220 (1980).

[0093] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103,Academic Press, 1996). Suitable culture media for this purpose include,for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

[0094] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0095] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by covalently joining to the immunoglobulin coding sequence allor part of the coding sequence for a non-immunoglobulin polypeptide. Inthat manner, “chimeric” or “hybrid” antibodies are prepared that havethe binding specificity of an anti-MUC18 monoclonal antibody herein.

[0096] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for an MUC18and another antigen-combining site having specificity for a differentantigen.

[0097] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0098] (b) Human Antibodies

[0099] Attempts to use the same technology for generating human mAbshave been hampered by the lack of a suitable human myeloma cell line.The best results were obtained using heteromyelomas (mouse×human hybridmyelomas) as fusion partners (Kozbor, J. Immunol. 133: 3001 (1984);Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63, Marcel Dekker, Inc., New York, 1987).Alternatively, human antibody-secreting cells can be immortalized byinfection with the Epstein-Barr virus (EBV). However, EBV-infected cellsare difficult to clone and usually produce only relatively low yields ofimmunoglobulin (James and Bell, J. Immunol. Methods 100: 5-40 [1987]).In the future, the immortalization of human B cells might possibly beachieved by introducing a defined combination of transforming genes.Such a possibility is highlighted by a recent demonstration that theexpression of the telomerase catalytic subunit together with the SV40large T oncoprotein and an oncogenic allele of H-ras resulted in thetumorigenic conversion of normal human epithelial and fibroblast cells(Hahn et al., Nature 400: 464-468 [1999]).

[0100] It is now possible to produce transgenic animals (e.g. mice) thatare capable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production(Jakobovits et al., Nature 362: 255-258 [1993]; Lonberg and Huszar, Int.Rev. Immunol. 13: 65-93 [1995]; Fishwild et al., Nat. Biotechnol. 14:845-851 [1996]; Mendez et al., Nat. Genet. 15: 146-156 [1997]; Green, J.Immunol. Methods 231: 11-23 [1999]; Tomizuka et al., Proc. Natl. Acad.Sci. USA 97: 722-727 [2000]; reviewed in Little et al., Immunol. Today21: 364-370 [2000]). For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production (Jakobovits et al., Proc.Natl. Acad. Sci. USA 90: 2551-2555 [1993]). Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant miceresults in the production of human antibodies upon antigen challenge(Jakobovits et al., Nature 362: 255-258 [1993]).

[0101] Mendez et al. (Nature Genetics 15: 146-156 [1997]) have generateda line of transgenic mice designated as “XenoMouse® II” that, whenchallenged with an antigen, generates high affinity fully humanantibodies. This was achieved by germ-line integration of megabase humanheavy chain and light chain loci into mice with deletion into endogenousJ_(H) segment as described above. The XenoMouse® II harbors 1,020 kb ofhuman heavy chain locus containing approximately 66 V_(H) genes,complete D_(H) and J_(H) regions and three different constant regions(μ, δ and γ), and also harbors 800 kb of human κ locus containing 32 Vκgenes, Jκ segments and Cκ genes. The antibodies produced in these miceclosely resemble that seen in humans in all respects, including generearrangement, assembly, and repertoire. The human antibodies arepreferentially expressed over endogenous antibodies due to deletion inendogenous J_(H) segment that prevents gene rearrangement in the murinelocus.

[0102] Techniques for generating antibodies using Abgenix's XenoMouse®technology include injection of a particular antigen of interest intosuch mice. Sera from such immunized animals may be screened forantibody-reactivity against the initial antigen. Lymphocytes may beisolated from lymph nodes or spleen cells and may further be selectedfor B cells by selecting for CD138-negative and CD19+ cells. The B cellcultures (BCCs) may be either fused to myeloma cells to generatehybridomas as detailed above or screened further for reactivity againstthe initial antigen. Such screening includes ELISA.

[0103] Transfection refers to the taking up of an expression vector by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ precipitation and electroporation.Successful transfection is generally recognized when any indication ofthe operation of this vector occurs within the host cell.

[0104] In a preferred embodiment, the antibodies of the presentinvention comprise an anti-human MUC18 monoclonal antibody heavy chainor a fragment thereof, comprising the following CDR's (as defined byKabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols 1-3): (a)CDR1, (b) CDR2 and (c) CDR3.

[0105] The heavy chain of the antibodies of one embodiment of thepresent invention comprise of the following sequences:

[0106] SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ IDNO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, ORSEQ ID NO: 37.

[0107] In yet another embodiment, the invention provides an anti-humanMUC18 monoclonal antibody light chain or a fragment thereof, comprisingthe following CDRs: (a) CDR1, (b) CDR2 and (c) CDR3. The light chain ofthe antibodies in one embodiment of the present invention comprise ofthe following sequences:

[0108] SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10; SEQ ID NO: 14, SEQ IDNO: 18; SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 30; SEQ ID NO: 34; orSEQ ID NO: 38.

[0109] In one aspect, the present invention includes anti-MUC18antibodies such as c3.19.1 and c6.11.3. The heavy chain amino acid andnucleotide sequences of c3.19.1 are encoded by SEQ ID NO: 1 and 3,respectively, and the heavy chain amino acid and nucleotide sequence ofc6.11.3 are encoded by 5 and 7, respectively. The light chain amino acidand nucleotide sequences of c3.1.9.1. are encoded by SEQ ID NO: 2 and 4,respectively, and the light chain amino acid and nucleotide sequences ofc6.11.3 are encoded by 6 and 8, respectively.

[0110] 2. Screening for Antibodies with the Desired Properties

[0111] Techniques for generating antibodies have been described above.One may further select antibodies with certain biologicalcharacteristics, as desired.

[0112] In one embodiment of the invention, melanoma cells were used foranalysis of anti-MUC18 antibody function. The melanoma cells werederived from a number of sources and possessed a variety ofcharacteristics that could potentially contribute to the metastaticphenotype. Their phenotypes are presented in Table 1. TABLE 1 Propertiesof Melanoma Cells Used for Studies Metastatic MUC18 Cell Line OriginCapacity Expression A375-SM Selection fromanimal High High model ofmetastasis SB-2 Cutaneous melanoma Non-metastatic Negative TXM-13 Brainmetastasis High High WM-2664 Metastasis from High Medium cutaneousmelanoma

[0113] (a) Binding to MUC18 Antigen

[0114] For example, to identify anti-MUC18 antibodies with high affinityfor human MUC18, kinetic measurements and binding affinity of theanti-MUC18 antibodies were obtained from Biacore experiments. TheBiacore experiments measured the affinity of MUC18 antibodies capturedon a protein A surface for labeled MUC18 antigen and are furtherdescribed in the examples below. Anti-MUC18 antibodies with a Kd of6×10⁻¹⁰ M were considered high affinity anti-MUC18 antibodies.

[0115] In a further example, to determine whether anti-MUC18 antibodiesof the present invention were able to recognize denatured MUC18 in humanmelanoma cells, the antibodies were used for immunoblots of metastaticmelanoma cells and non-metastatic melanoma cells (control). Thoseantibodies which were able to detect MUC18 in metastatic melanoma cellswere selected as anti-MUC18 antibodies of interest.

[0116] Further, to identify anti-MUC18 antibodies that recognized thenative form of the MUC18 protein on the surface of cells, flow cytometryanalysis was performed. According to this assay, cells expressing theantigen of interest were detached from cell culture plates, incubatedwith either an isotype-matched control human antibody or the anti-MUC18antibody for 20 minutes at 4° C. After washing, all samples wereincubated with phycoerythrin-conjugated F(ab′)₂ fragments of GoatAnti-Human IgG (H+L) (Jackson) for 20 minutes at 4° C. in the dark.After several washings, the cells were resuspended in FACS buffer andanalyzed by cytofluorometry. Those antibodies which shift thefluorescence intensity when compared to control antibodies were selectedas anti-MUC18 antibodies of interest.

[0117] (b) Inhibition of Metastasis

[0118] To select for metastasis inhibitory anti-MUC18 antibodies,antibodies were screened for the ability to inhibit metastasis in animalmodels. Exponentially growing A375SM cells were harvested on Day 0,resuspended and injected into the lateral tail veins of female nudemice. The animals were treated one day prior to tumor cell injection andonce a week thereafter with a specific dose of anti-MUC18 antibody. Allanimals were sacrificed after 6 weeks at which time the lungs wereremoved and the tumor nodules were counted with the aid of a dissectingmicroscope. Those antibodies which inhibited melanoma metastasis intothe lung in a dose-dependent manner were selected as metastasisinhibitory antibodies.

[0119] (c) Promotion of Animal Survival

[0120] To select for antibodies which increase the survival of animalsbearing metastatic melanoma tumors, antibodies were screened for theability to increase the survival of mice bearing tumors. WM-2664 humanmelanoma tumor cells in their exponential phase were harvested,resuspended in suitable buffer, and injected into the lateral tail veinof male BALB/c nude mice. After administration of anti-MUC18 antibody orcontrol antibody intraperitoneally at 1 mg or 0.2 mg per mouse on aweekly basis, mice were monitored daily for survival. Those antibodieswhich demonstrated a dose dependent increase in survival of the micewere selected as survival prolonging antibodies.

[0121] 3. Therapeutic Compositions and Mode of Administration ofAnti-MUC18 Antibodies

[0122] Therapeutic formulations of the anti-MUC18 antibodies of theinvention are prepared for storage by mixing antibody having the desireddegree of purity with optional physiologically acceptable carriers,excipients, or stabilizers (Remington: The Science and Practice ofPharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton,Pa.: 1995)), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

[0123] The anti-MUC18 antibody to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution. The anti-MUC18 antibody ordinarily will be stored inlyophilized form or in solution.

[0124] Therapeutic anti-MUC18 antibody compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0125] The route of anti-MUC18 antibody administration is in accord withknown methods, e.g. injection or infusion by intravenous,intraperitoneal, intracerebral, subcutaneous, intramuscular,intraocular, intraarterial, intracerebrospinal, or intralesional routes,or by sustained release systems as noted below. Preferably the antibodyis given systemically.

[0126] Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer,Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release anti-MUC18 antibody compositions may also includeliposomally entrapped antibody. Liposomes containing antibody areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal antibody therapy.

[0127] Anti-MUC18 antibody can also be administered by inhalation.Commercially available nebulizers for liquid formulations, including jetnebulizers and ultrasonic nebulizers are useful for administration.Liquid formulations can be directly nebulized and lyophilized powder canbe nebulized after reconstitution. Alternatively, anti-MUC18 antibodycan be aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

[0128] An “effective amount” of anti-MUC18 antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, the type of anti-MUC18 antibodyemployed, and the condition of the patient. Accordingly, it will benecessary for the therapist to titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the anti-MUC18 antibody until adosage is reached that achieves the desired effect. The progress of thistherapy is easily monitored by conventional assays.

[0129] Antibodies specific to tumor antigens such as anti-MUC18 areuseful in targeting of tumor cells for destruction. For example, ricin,a cellular toxin derived from plants, is finding unique applications,especially in the fight against tumors and cancer. Implications arebeing discovered as to the use of ricin in the treatment of tumors.Ricin has been suggested to have a greater affinity for cancerous cellsthan normal cells (Montfort et al. 1987) and has been often termed as a“magic bullet” for targeting malignant tumors. Toxins such as ricinremain active even if the B chain of the toxin is removed. Accordingly,if the solitary A chain is coupled to a tumor-specific antibody, such asanti-MUC18 antibody, the toxin has a specific affinity for cancerouscells over normal cells (Taylorson 1996). For example, ricin immunotoxinhas been developed to target the CD5 T-cell antigen often found inT-cell and B-cell malignancies (Kreitman et al. 1998). Further, thelinking of such anti-MUC18 antibodies to radioisotopes providesadvantages to tumor treatments. Unlike chemotherapy and other forms ofcancer treatment, radioimmunotherapy or the administration of aradioisotope-antibody combination directly targets the cancer cells withminimal damage to surrounding normal, healthy tissue. With this “magicbullet,” the patient can be treated with much smaller quantities ofradioisotopes than other forms of treatment available today. Mostcommonly antibodies are conjugated with potent chemotherapeutic agentssuch as maytansine, geldanamycin or calichaemycin for delivery to tumors(Frankel et al., Cancer Biotherapy and Radiopharmaceuticals, 15:459-476(2000); Knoll et al., Cancer Res., 60:6089-6094 (2000); Liu et al.,Proc. Natl. Acad. Sci. USA, 93:8618-8623 (1996); Mandler et al., J.Natl. Cancer Inst., 92:1573-1581 (2000); and Ota et al., Int. J. Clin.Oncol., 4:236-240 (1999). These drugs are too toxic to be administeredon their own. When conjugated to a therapeutic antibody such as MUC18,their biological activity can be directed specifically to the tumorcells. Accordingly, antibodies, such as MUC18 antibodies, can bemodified to act as immunotoxins utilizing techniques that are well knownin the art. See e.g., Vitetta et al., Immunol. Today, 14:252 (1993) andU.S. Pat. No. 5,194,594. In connection with the preparation ofradiolabeled antibodies, such modified antibodies can also be readilyprepared utilizing techniques that are well known in the art. See e.g.,Junghans et al., Cancer Chemotherapy and Biotherapy, pgs. 655-686(second edition, Chafner and Longo, eds., Lippincott Raven (1996)) andU.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990, 5,648,471,and 5,697,901. The immunotoxins and radiolabeled molecules would belikely to kill cells expressing MUC18, and particularly those cells inwhich the antibodies of the invention are effective.

[0130] The patients to be treated with the anti-MUC18 antibody of theinvention include patients with tumors, preferably melanomaand/orprostate or renal cancer. Other tumors include esophageal, pancreatic,colorectal tumors, carcinomas, such as renal cell carcinoma (RCC),cervical carcinomas and cervical intraepithelial squamous and glandularneoplasia, and cancers, such as colorectal cancer, breast cancer, lungcancer, and other malignancies. Patients are candidates for therapy inaccord with this invention until such point as no healthy tissue remainsto be protected from tumor progression. It is desirable to administer ananti-MUC18 antibody as early as possible in the development of thetumor, and to continue treatment for as long as is necessary.

[0131] In the treatment and prevention of tumor-associated disorder byan anti-MUC18 antibody, the antibody composition will be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the antibody, the particular type ofantibody, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of antibody to be administered willbe governed by such considerations, and is the minimum amount necessaryto prevent, ameliorate, or treat the disorder, including treatingchronic autoimmune conditions and immunosuppression maintenance intransplant recipients. Such amount is preferably below the amount thatis toxic to the host or renders the host significantly more susceptibleto infections.

[0132] As a general proposition, the initial pharmaceutically effectiveamount of the antibody administered parenterally will be in the range ofabout 0.1 to 50 mg/kg of patient body weight per day, with the typicalinitial range of antibody used being 0.3 to 20 mg/kg/day, morepreferably 0.3 to 15 mg/kg/day. The desired dosage can be delivered by asingle bolus administration, by multiple bolus administrations, or bycontinuous infusion administration of antibody, depending on the patternof pharmacokinetic decay that the practitioner wishes to achieve.

[0133] As noted above, however, these suggested amounts of antibody aresubject to a great deal of therapeutic discretion. The key factor inselecting an appropriate dose and scheduling is the result obtained, asindicated above. For example, the antibody may be optionally formulatedwith one or more agents currently used to prevent or treat tumors suchas standard- or high-dose chemotherapy and hematopoietic stem-celltransplantation. The effective amount of such other agents depends onthe amount of anti-MUC18 antibody present in the formulation, the typeof disorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

[0134] Further details of the invention can be found in the followingexample, which further defines the scope of the invention. Allreferences cited throughout the specification, and the references citedtherein, are hereby expressly incorporated by reference in theirentirety.

EXAMPLE 1

[0135] Preparation of MUC18 Antigens

[0136] In the present study, recombinant MUC18 proteins were prepared.The extracellular domain (ECD) (aa#1-559) of human MUC18 was cloned fromSK-MEL-28 cells (ATCC HTB-72) by Reverse Transcriptase-PCR (RT-PCR) withprimers that incorporate an EcoRI site in the forward primer and an NheIsite in the reverse primer based on the published NCBI sequence(Accession # NM_(—)006500).

[0137] The primers used for the amplification of the ECD of MUC18 wereas follows: Forward primer: 5′-ATATTACGAATTCACTTGCGTCTCGCCCTCCGG-3′ (SEQID NO: 10) Reverse primer: 5′-CAGCTTAGAGCTAGCCGGCTCTCCGGCTCCGGCA-3′ (SEQID NO: 11)

[0138] MUC18 cDNA was amplified (Gene Amp XL PCR kit, Perkin Elmer) fromRNA (RNAzol,Tel Test, INC) prepared from SK-MEL-28 cells (ATCC HTB-72).For construction of a V5-HIS or HuIgG2 fusion protein, the 1700 bp PCRproduct encoding amino acids 1-559 was digested with EcoRI and NheI andligated into CD147HuIgG2DHFR vector (ABGX) digested with EcoRI and NheIor pcDNA3.1V5HISB vector (Invitrogen) digested with EcoRI and XbaI. Theresulting plasmids were transfected in 293 cells by CaPO₄ method, andthen, the fusion protein was purified from harvested conditioned mediavia ProteinA chromatography (MUC18-HuIgG2) or Ni—NTA chromatography(MUC18-V5HIS).

[0139] The MUC18 ECD contained 4 amino acid differences from thepublished NCBI sequence: #383 D>G,#390 P>L,#424 K>N, and #425 L>V.

EXAMPLE 2

[0140] Anti-MUC18 Antibodies

[0141] A. Antibody Generation

[0142] 1. Immunization and Selection of Animals for Harvesting by ELISA

[0143] Monoclonal antibody against MUC18 was developed by sequentiallyimmunizing XenoMouse mice (XenoMouse G2, Abgenix, Inc. Fremont, Calif.).The initial immunization was with 5×10⁶ SK-MEL-28 cells admixed 1:1 v/vwith Complete Freund's Adjuvant (CFA). Subsequent boosts were made firstwith 5×10⁶ SK-MEL-28 cells admixed 1:1 v/v with Incomplete Freund'sAdjuvant (IFA), followed by four injections with 5 μg solubleMUC18-human IgG₂ Fe fusion protein admixed 1:1 v/v with IFA, and then afinal boost of 10 μg soluble MUC18-human IgG2 Fe fusion protein withoutadjuvant. In particular, each mouse was immunized either at the base ofthe tail by intraperitoneal injection or via hind footpad injection withMUC18 recombinant antigen followed by the generation of a large numberof candidate mAbs, and the screening of antibodies for binding andactivity.

[0144] The mice were initially injected with MUC18 antigen at aconcentration of 1-5 μg/mouse. Each mouse was further immunized intoeach hind footpad 6 additional times (at 3-4 day intervals) with solubleantigen, specifically 5 μg of soluble MUC18-human IgG2 Fc fusion proteinin DPBS admixed 1:1 v/v with IFA then a final boost of 10 μg solubleMUC18-human IgG2 Fe fusion protein in DPBS without adjuvant. The animalswere immunized on days 0, 4, 7, 10, 14, 17 and 20 and four days later onday 4, fusions were performed. For the fusions, the mice wereeuthanized, and inguinal and popliteal lymph nodes were recovered.

[0145] Lymphocytes from the immunized XenoMouse mice were released bymechanical disruption of the lymph nodes using a tissue grinder and thendepleted of T cells by CD90 negative selection. The fusion was performedby mixing washed enriched B cells and non-secretory myeloma P2X63Ag8.653cells purchased from ATCC (Cat. #CRL 1580) (Kearney et al., J. Immunol.,123:1548-1550 (1979)) at a ratio of 1:1. The cell mixture was gentlysubjected to centrifugation at 800 g. After complete removal of thesupernatant, the cells were treated with 2-4 mL of Pronase solution(CalBiochem, Cat. #53702; 0.5 mg/mL in PBS) for no more than 2 minutes.Then 3-5 ml of FBS was added to stop the enzyme activity, and thesuspension was adjusted to 40 mL total volume using electro cell fusionsolution, ECFS (0.3M Sucrose, Sigma, Cat#S7903; 0.1 mM MagnesiumAcetate, Sigma, Cat. #M2545; 0.1 mM Calcium Acetate, Sigma, Cat#C4705).The supernatant was removed after centrifugation and the cells wereresuspended in 40 mL ECFS. This wash step was repeated, and the cellsagain were resuspended in ECFS to a concentration of 2×106 cells/mL.Electro-cell fusion was performed using a fusion generator, modelECM2001, Genetronic, Inc., San Diego, Calif.

[0146] After fusion, the cells were resuspended in DMEM (JRHBiosciences), 15% FCS (Hyclone), containing HAT, and supplemented withL-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin)(all from Sigma) and IL-6 (Boehringer Mannheim) for culture at 37° C.and 10% CO₂ in air. Cells were plated in flat-bottomed 96-well tissueculture plates at 4×10⁴ cells per well. Cultures were maintained in HAT(hypoxanthine, aminopterin and thymidine) supplemented media for 2 weeksbefore transfer to HT (hypoxanthine and thymidine) supplemented media.Hybridomas were selected for by survival in HAT medium and supernatantsfrom those wells containing hybridomas were screened for antigenreactivity by ELISA. The ELISA format entailed incubating supernatantson antigen coated plates and detecting human anti-MUC18 binding usinghorseradish peroxidase (HRP) labeled mouse anti-human IgG2.

[0147] Cloning was performed on selected antigen-positive wells usinglimited dilution plating. Plates were visually inspected for thepresence of single colony growth and supernatants from single colonywells then screened by antigen-specific ELISA as described above. Highlyreactive clones were assayed to verify purity of human gamma and kappachain by multiplex ELISA using a Luminex instrument.

[0148] Based on the assay results, the following clones were identifiedas anti-MUC18 antibodies: c3.19.1, c6.11.3, c3.10, c3.22, c3.27, c3.45,c3.65, c6.1, c6.9, c6.2, and c6.12. c6.9 and c6.12 were identicalindividually identified clones. The antibodies of the present inventionwere analyzed for sequence similarity to germline V_(H) and V_(K) genes.Such analysis is summarized in Table 2 and FIG. 35. The amino acidsequences of the heavy and light chain variable regions of the MUC18antibodies of the present invention were further aligned with germlineV_(H) and V_(K) sequences, respectively. These alignments are shown inFIGS. 15-16 (c3.10), FIGS. 17-18 (C3.22), FIGS. 19-20 (C3.27), FIGS.21-22 (c3.45), FIGS. 23-24 (c3.65), FIGS. 25-26 (c6.1), FIGS. 27-28(c6.12), FIGS. 29-30 (c6.2), FIGS. 31-32 (c6.9), and FIGS. 33-34 (c6.11). c3.19.1 was selected for further characterization. TABLE 2Comparison of CDR regions in MUC18 antibody clones with CDR regions ingermline V_(H) and V_(K) genes No. of Nucleotide/Amino acid changesClone Germline genes used FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 MUC18 V D J V D& J A15-3.10 VH V4-59 D21-9 JH3B 0/0 0/0 1/0 3/3 5/2 0/0 0/0 VK 02 JK20/0 1/1 1/0 1/1 1/0 1/1 0/0 A15-3.22 VH V4-31 — JH4B 0/0 2/1 0/0 1/1 0/00/0 0/0 VK A30 JK4 0/0 0/0 0/0 0/0 0/0 0/0 0/0 A15-3.27 VH V4-59 D21-9JH3B 0/0 0/0 1/0 4/4 6/1 0/0 0/0 VK A30 JK1 0/0 0/0 0/0 0/0 1/0 0/0 0/0A15-3.45 VH V1-18 D3-10 JH6B 1/0 4/2 1/1 0/0 1/0 0/0 0/0 VK B3 JK1 2/12/2 0/0 0/0 2/2 0/0 0/0 A15-3.65 VH 4-31 D6-13 JH5A 0/0 2/2 0/0 1/1 4/40/0 0/0 VK 08 JK4 0/0 1/1 0/0 0/0 2/1 1/1 0/0 A15-6.1 VH V3-30 D3-3 JH6B1/0 1/1 0/0 0/0 0/0 0/0 0/0 VK A20 JK3 0/0 1/1 1/1 1/1 0/0 3/3 2/1A15-6.2 VH V4-59 D6-19 JH3B 1/0 2/1 1/0 4/3 4/2 0/0 0/0 VK A19 JK4 2/12/2 0/0 0/0 2/2 2/1 0/0 A15-6.9 VH V4-31 D5-24 JHI 4/3 3/2 2/1 1/1 2/10/0 0/0 VK L2 JK1 0/0 3/3 1/1 0/0 2/0 0/0 0/0 A15-6.11 VH V4-31 D5-24JH1 0/0 3/2 0/0 0/0 0/0 0/0 0/0 VK L2 JK1 0/0 2/1 0/0 0/0 0/0 0/0 0/0A15-6.12 VH V4-31 D5-24 JH1 4/3 3/2 2/1 1/1 2/1 0/0 0/0 VK L2 JK1 0/03/3 1/1 0/0 2/0 0/0 0/0

[0149] B. Characterization of MUC18 Antibodies

[0150] 1. Binding of Anti-MUC18 Antibodies to MUC18 Antigen

[0151] (a) Immunoblot Analysis of Binding of Anti-MUC18 Antibody toMUC18

[0152] To determine whether anti-MUC18 antibody recognized MUC18expressed on melanoma cell lines, melanoma cell lines A375SM, SB2,TXM-13, WM-2664 and nude mouse endothelial cells (NME) were seeded(1×10⁶) in 100 mm tissue culture plates (Falcon) in 10 mL completegrowth medium. After overnight incubation, the plates were washed twotimes in PBS, and scraped in 400 μl Triton lysis buffer containing acocktail of protease inhibitors plus DTT. Following centrifugation, theprotein concentration was determined using a kit from BioRad. 40 μg ofprotein was loaded onto a 10% SDS-PAGE and electrophoreticallytransferred to a 0.45 -micron nitrocellulose membrane (Millipore). Themembrane was incubated in buffer containing anti-MUC18 antibodyovernight, reacted with a conjugated secondary antibody (Anti-human IgG)for one hour and the proteins were subsequently detected by the ECL(Amersham Corp) method according to the manufactures protocol.

[0153] Anti-MUC18 antibodies detected high levels of MUC18 in themetastatic A375SM, TXM-13 and WM-2664 cells and no signal in thenonmetastatic cell line SB-2 and normal mouse endothelial (NME2) cellswere MUC18 (FIG. 2). The reason for the lack of a signal in the NME2 inthis experiment was due most likely to the failure of anti-MUC18antibody to cross-react with mouse MUC18 protein.

[0154] Further, these results corroborate the findings of others withrespect to a positive correlation between MUC18 expression and themetastatic capacity of melanoma cells (Shih et al., Clinical CancerRes., 2:569-575 (1996); Johnson et al., Cancer Metastasis Rev.,18:345-357 (1999); Xie et al., Oncogene, 15(17):2069-75 (1997); Xie etal., Cancer Res., 57(11):2295-303 (1997); Schlagbauer-Wadl et al., Int JCancer, 81(6):951-5 (1999)).

[0155] (b) Flow Cytometric Analysis of Binding of Anti-MUC18 Antibody toMUC18

[0156] To determine whether anti-MUC18 antibody recognized the nativeform of the MUC18 protein on the surface of cells, flow cytometryanalysis was performed.

[0157] A375-SM and WM-2664 cells (4×10⁵) were detached with PBS-EDTA andincubated in FACS buffer (PBS, 2% FBS and 0.02% sodium azide) witheither an isotype-matched control human IgG2 antibody or anti-MUC18antibody for 20 minutes at 4° C. After washing with FACS buffer, allsamples were incubated with phycoerythrin-conjugated F(ab′)₂ fragmentsof Goat Anti-Human IgG (H+L) (Jackson) for 20 minutes at 4° C. in thedark. After several washings, the cells were resuspended in FACS bufferand analyzed by cytofluorometry.

[0158] As shown in FIG. 3, neither the A375-SM nor the WM-2664 cellsdemonstrated a fluorescent shift when incubated in the presence of thecontrol IgG2 Ab (bold line). However, when incubated in the presence ofanti-MUC18 (dotted line), a strong shift in fluorescence intensityindicative of cell surface expression of the antigen was observed. Theseresults show that anti-MUC18 antibody can recognize the native MUC18antigen expressed on the surface of human melanoma cells.

[0159] (c) Binding Kinetics and Affinity of MUC18 to Anti-MUC18 Antibody

[0160] A Biacore 3000 instrument was used for all kinetic measurementswith HBS-P (Hepes-buffered saline, 0.005% polysorbate 20) buffer. Themeasurements were made utilizing three B1 sensor chips(carboxymethyldextran matrix with a low amount of carboxylation). Theexperiments were performed by covalently immobilizing protein A bystandard amine coupling at a level of 1500-3000 RU (resonance units) onthe surface of the four flow cells of a B1 chip. MAb 3.19.1 was capturedby flowing a 1 μg/ml solution of 3.19.1 at a flow rate of 60 μL/min. for20-30 sec. across the protein A surface, giving a captured level of110-250 RU. The control protein A surface did not have any MAb capturedon it. Various concentrations of MUC18-V5-His antigen, ranging from 0.5nM-100 nM, were flowed across the surface in triplicate for 2.5 minutesat 100 μL/min., and the dissociation phase was followed for 10 mins. Thedata were processed by “Scrubber”, version 1.10, and the processedsensorgrams were non-linearly fit by “Clamp”, version 3.40, employing asimple bimolecular 1:1 kinetic model (Table 3). TABLE 3 Binding Kineticsand Affinity of anti-MUC18 antibody (c3.19.1) for MUC18 Antigen Date ofChip Measure- Desig- c3.19.1 ment nation* Lot# k_(a) (M⁻¹s⁻¹) k_(d)(s⁻¹) K_(d) (nM) May 2001  I 385020A 4.531 × 10⁵ 3.021 × 10⁴ 0.67October  II 360-67 7.090 × 10⁵ 4.019 × 10⁴ 0.57 2001 October  II 360-675.746 × 10⁵ 3.961 × 10⁴ 0.69 2001 November III 360-67 7.494 × 10⁵ 3.466× 10⁴ 0.46 2001 November III 360-67 6.251 × 10⁵ 3.852 × 10⁴ 0.62 2001November III RD#1 6.146 × 10⁵ 4.021 × 10⁴ 0.65 2001 November III RD#26.608 × 10⁵ 3.894 × 10⁴ 0.59 2001 95% Confidence Average StandardDeviation Interval k_(a) (M⁻¹s⁻¹) 6.27 × 10⁵ ±9.66 × 10⁴ ±8.94 × 10⁴(14%) k_(d) (s⁻¹) 3.75 × 10⁻⁴ ±3.73 × 10⁻⁵ ±3.45 × 10⁻⁵ (9.2%) K_(d)(nM) 0.61 ±0.078 ±0.072 (12%)

[0161] 2. Effect of Anti-MUC18 Antibody on Metastasis of Melanoma Cellsin vivo

[0162] Because MUC18 expression is most closely associated with themetastatic phenotype in melanoma patients, the ability of anti-MUC18antibody (c3.19.1) to inhibit the formation of lung metastases wheninjected intravenously into the tail veins of nude mice was examined.Exponentially growing A375-SM cells were harvested on Day 0, resuspendedin Hanks' balanced salt solution (HBSS) and 4×10⁵ viable tumor cellswere injected into the lateral tail veins of female nude mice fromHarlan Laboratories. The animals were treated one day prior to tumorcell injection and once a week thereafter with the indicated dose ofanti-MUC18 antibody. All animals were sacrificed after six weeks atwhich time the lungs were removed and the tumor nodules counted with theaid of a dissecting microscope. The results of this experiment arepresented in Table 4 and demonstrate that anti-MUC18 antibody inhibitslung tumor formation in a dose-dependent manner. The total number oflung metastases was decreased in all treated animals. In mice receivingthe 1.0 mg per mouse dose of anti-MUC18 (c3.19.1), the tumor burden wasvery low and no animals had more than 50 nodules in their lungs. TABLE 4A375-SM Melanoma Metastasis Formation in Mouse Lungs Animals AnimalsAnimals with ≦10 with 11-50 with >50 Median No. Treatment Incidence ofTumors Tumors Tumors Tumors Tumors/Animal Control 13/14  7/14 3/14 4/1410 Antibody c3.19.1 10/14  6/14 5/14 3/14 12 0.1 mg/dose c3.19.1 14/1412/14 2/14 0/14 5 1.0 mg/dose

[0163] One additional study was performed in vivo to evaluate theability of anti-MUC18 antibody (c3.19.1) to increase the survival ofmice bearing metastatic melanoma tumors. The WM-2664 human melanomatumor cells in their exponential growth phase were harvested andresuspended in PBS. Viable tumor cells (10⁶ in 0.2 mL PBS) were injectedinto the lateral tail vein of male BALB/c nude mice on Day 0. On thesame day, the animals were administered PBS (n=21), c3.19.1 (n=12) orisotype-matched control IgG2 antibody (n=12) intraperitoneally at 1 mgor 0.2 mg per mouse, and once weekly thereafter. The mice were monitoredeveryday for survival. Autopsies were performed on dead mice from thedifferent groups to confirm the presence of tumor metastases. The datawere expressed as percent survival calculated as follows:100−[100×(Number of dead mice/Total number of mice)].

[0164]FIG. 4 demonstrated that treatment with anti-MUC18 antibody(c3.19.1) can prolong the survival of mice bearing metastatic melanomatumors. A dose dependent increase in survival was observed and to dateno animals in the group receiving the high dose of anti-MUC18 antibodyhave died due to tumor burden.

[0165] The role of MUC18 in melanoma tumor progression and the mechanismof anti-MUC18 antibody (c3.19.1) action on this target is not completelyunderstood. Although anti-MUC18 antibody does not inhibit the growth ofmelanoma tumor cells in cell culture, it does inhibit the growth ofsubcutaneous and metastatic tumor cells in vivo. The cumulative evidenceindicates that MUC18 plays a role in one or more steps in the metastaticprocess possibly by affecting MMP-2 activation or cell migration. Whenconsidered together these data provide evidence that anti-MUC18 antibodyis a promising therapeutic antibody for inhibiting the growth andmetastasis of human melanoma cells in patients with this disease.

EXAMPLE 3

[0166] Antibody Conjugates

[0167] Antibodies specific to antigens such as anti-MUC18 are useful intargeting of tumor cells expressing such antigens for elimination.

[0168] A. Linkage of Anti-MUC18 Antibody to Ricin

[0169] Ricin, a cellular toxin, is finding unique applications,especially in the fight against tumors and cancer. Implications arebeing discovered as to the use of ricin in the treatment of tumors.Ricin has been suggested to have a greater affinity for cancerous cellsthan normal cells (Montfort et al. 1987) and has been often termed as a“magic bullet” for targeting malignant tumors. Toxins such as ricinremain active even if the B chain which is responsible for because oftoxin nonspecific lectin activity leads to toxic side effects isremoved. Accordingly, if the solitary A chain is coupled to atumor-specific antibody, the toxin has a specific affinity for cancerouscells over normal cells (Taylorson 1996). For example, ricin immunotoxinhas been developed to target the CD5 T-cell antigen often found inT-cell and B-cell malignancies (Kreitman et al. 1998).

[0170] A novel method of coupling whole intact ricin to monoclonalantibody is described in Pietersz et al. (Cancer Res 48(16):4469-76(1998)) and includes blocking of nonspecific binding of the ricinB-chain. Coupling of ricin to the anti-MUC18 antibodies of the presentinvention may be done by using the bifunctional reagentsS-acetylmercaptosuccinic anhydride for antibody and succinimidyl3-(2-pyridyldithio)propionate for ricin. The coupling should result inthe loss of B-chain binding activity, while impairing neither the toxicpotential of the A-chain nor the activity of the antibody. Wholericin-antibody conjugates produced in this way should not bindnonspecifically to target cells, the most important implication beingthat such immunotoxins should be more potent that ricin A-chainconjugates and capable of being used in vivo.

[0171] B. Linkage to Radioisotope

[0172] The linking of such anti-MUC18 antibodies to radioisotopesprovides advantages to tumor treatments. Unlike chemotherapy and otherforms of cancer treatment, radioimmunotherapy or the administration of aradioisotope-antibody combination directly targets the cancer cells withminimal damage to surrounding normal, healthy tissue. With this “magicbullet,” the patient can be treated with much smaller quantities ofradioisotopes than other forms of treatment available today. Preferredradioisotopes include yttrium⁹⁰ (90Y), indium¹¹¹ (111In), ¹³¹I, ⁹⁹mTc,radiosilver-111, radiosilver-199, and Bismuth²¹³.

[0173] Linkage of radioisotopes to antibodies may be performed withconventional bifunction chelates. Since silver is monovalent, forradiosilver-111 and radiosilver-199 linkage, sulfur-based linkers may beused (Hazra et al., Cell Biophys, 24-25:1-7 (1994)). Linkage of silverradioisotopes may involved reducing the immunoglobulin with ascorbicacid. In another aspect, tiuxetan is an MX-DTPA linker chelator attachedto ibritumomab to form ibritumomab tiuxetan (Zevalin) (Witzig, T. E,Cancer Chemother Pharmacol, 48 Suppl 1:S91-5 (2001). Ibritumomabtiuxetan can react with radioisotypes such as indium¹¹¹ (111In) or 90Yto form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan,respectively.

[0174] C. Linkage of Anti-MUC18 Antibody to Toxic ChemotherapeuticAgents

[0175] Most commonly antibodies to treat cancer are being conjugatedwith toxic chemotherapeutic drugs such as maytansine, geldanamycin orcalichaemycin. Different linkers that release the drugs under acidic orreducing conditions or upon exposure to specific proteases are employedwith this technology.

EXAMPLE 4

[0176] Uses of Anti-MUC18 Antibodies and Antibody Conjugate

[0177] A. Treatment of humans with anti-MUC18 antibodies

[0178] To determine the in vivo effects of anti-MUC18 antibody treatmentin human patients with tumors, such human patients are injected over acertain amount of time with an effective amount of anti-MUC18 antibody.At periodic times during the treatment, the human patients are monitoredto determine whether their tumors progress, in particular, whether thetumors grow and metastasize.

[0179] A tumor patient treated with anti-MUC18 antibodies have a lowerlevel of tumor growth and metastasis compared to the level of tumorgrowth and metastasis of tumors in tumor patients treated with controlantibodies. Control antibodies that may be used include antibodies ofthe same isotype as the anti-MUC18 antibodies tested and further, maynot have the ability to bind to MUC18 tumor antigen.

[0180] B. Treatment with Anti-MUC18 Antibody Conjugates

[0181] To determine the in vivo effects of anti-MUC18 antibodyconjugates, human patients or animals exhibiting tumors are injectedover a certain amount of time with an effective amount of anti-MUC18antibody conjugate. In one embodiment, the anti-MUC18 antibody conjugateadministered is maytansine-anti-MUC18 antibody conjugate orradioisotope-anti-MUC18 antibody conjugate. At periodic times during thetreatment, the human patients or animals are monitored to determinewhether their tumors progress, in particular, whether the tumors growand metastasize.

[0182] A human patient or animal exhibiting tumors and undergoingtreatment with either maytansine-anti-MUC18 antibody orradioisotope-anti-MUC18 antibody conjugates have a lower level of tumorgrowth and metastasis when compared to a control patient or animalexhibiting tumors and undergoing treatment with control antibodyconjugates, such as control maytansine-antibody or controlradioisotope-antibody. Control maytansine-antibodies that may be usedinclude conjugates comprising maytansine linked to antibodies of thesame isotype of the anti-MUC18 antibodies, but more specifically, nothaving the ability to bind to MUC18 tumor antigen. Controlradioisotope-antibodies that may be used include conjugates comprisingradioisotope linked to antibodies of the same isotype of the anti-MUC18antibodies, but more specifically, not having the ability to bind toMUC18 tumor antigen.

[0183] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents.

1 40 1 121 PRT Homo Sapiens 1 Gln Val Gln Leu Gln Glu Ser Gly Pro GlyLeu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val SerGly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro ProGly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Thr Trp Thr SerAsn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp ThrSer Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Ser Ser Val Thr Ala AlaAsp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gln Gly Gln Trp Leu LeuPro Asp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr ValSer Ser 115 120 2 112 PRT Homo Sapiens 2 Asp Ile Val Met Thr Gln Ser ProLeu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser CysArg Ser Ser Gln Ser Leu Leu Arg Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu AspTrp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro His Leu Leu Ile Tyr LeuGly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala GluAsp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Gln Gln Ser Pro Ile ThrPhe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 3 364 DNA HomoSapiens 3 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagaccctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact ggagctggatccggcagccc 120 ccagggaagg gactggagtg gattggctat atctattaca cttggacctccaactacaac 180 ccctccctca agagtcgcgt caccatatca gtggacacgt ccaaaaaccagttctccctg 240 aggctgagtt ctgtgaccgc tgcggacacg gccgtttatt actgtgcgagagatcagggg 300 cagtggttac tacccgatgc ttttgatatc tggggccaag ggacaatggtcaccgtctct 360 tcag 364 4 337 DNA Homo Sapiens 4 gatattgtga tgactcagtctccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtcagagcctcctg cgtagtaatg gatacaacta tttggattgg 120 tacctgcaga agccaggacagtctccacat ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggttcagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgaggatgttggggtt tattactgca tgcaagctca acaaagtccg 300 atcaccttcg gccaagggacacgactggag attaaac 337 5 117 PRT Homo Sapiens 5 Gln Val Gln Leu Gln GluSer Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu ThrCys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Thr Tyr His Trp SerTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr IleTyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg ValThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys LeuSer Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg GlyGly Asp Gly Tyr Lys Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr ValSer Ser 115 6 107 PRT Homo Sapiens 6 Glu Ile Val Met Thr Gln Ser Pro AlaThr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys ArgAla Ser Gln Ser Val Ser Asn Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys ProGly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala ThrGly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe ThrLeu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr TyrCys Gln Gln Tyr Asn Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr LysVal Glu Ile Lys 100 105 7 352 DNA Homo Sapiens 7 caggtgcagc tgcaggagtcgggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtggctccatcagc agtggtactt accactggag ctggatccgc 120 cagcacccag ggaagggcctggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagagtcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgagctctgtgactgccgcg gacacggccg tgtattactg tgcgagaggg 300 ggagatggct acaagtactggggccaggga accctggtca ccgtctcctc ag 352 8 322 DNA Homo Sapiens 8gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc aacaacttag cctggtatca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact ggcctcggac gttcggccaa 300gggaccaagg tggaaatcaa ac 322 9 121 PRT Homo Sapiens 9 Gln Val Gln LeuGln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu SerLeu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp SerTrp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr IleTyr Tyr Thr Trp Thr Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg ValThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg LeuSer Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Tyr Cys Ala 85 90 95 Arg AspGln Gly Gln Trp Leu Leu Pro Asp Ala Phe Asp Ile Trp Gly 100 105 110 GlnGly Thr Met Val Thr Val Ser Ser 115 120 10 109 PRT Homo Sapiens 10 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580 Glu Asp Phe Ala Thr Tyr Tyr Cys Arg Gln Ser Tyr Ser Thr Pro Pro 85 9095 Glu Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 11 364DNA Homo Sapiens 11 caggtgcagc tgcaggagtc gggcccagga ctggtgaagccttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactactggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattggctat atctattacacttggaccac caactacaac 180 ccctccctca agagtcgcgt caccatatca gtggacacgtccaagaacca gttctccctg 240 aggctgagct ctgtgaccgc tgcggacacg gccctttattactgtgcgag agatcagggg 300 cagtggttac tacccgatgc ttttgatatc tggggccaagggacaatggt caccgtctct 360 tcag 364 12 328 DNA Homo Sapiens 12 gacatccagatgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgccgggcaagtca gagcattagc aactatttaa attggtatca gcagaaacca 120 ggaaaagcccctaagctcct gatctatggt gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtggcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttgcaacctacta ctgtcgacag agttacagta cccctccgga gtgcagtttt 300 ggccaggggaccaagctgga gatcaaac 328 13 117 PRT Homo Sapiens 13 Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser LeuThr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr TrpThr Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly PheIle Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser ArgVal Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu LysLeu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala ArgGlu Gly Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val ThrVal Ser Ser 115 14 107 PRT Homo Sapiens 14 Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser LeuGln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala ThrTyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly GlyThr Lys Val Glu Ile Lys 100 105 15 352 DNA Homo Sapiens 15 caggtgcagctgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactgtctctggtgg ctccatcagc agtggtggtt actactggac ttggatccgc 120 cagcacccagggaagggcct ggagtggatt gggttcatct attacagtgg gagcacctac 180 tacaacccgtccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaagctgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300 ggagatggctttgactactg gggccaggga accctggtca ccgtctcctc ag 352 16 322 DNA HomoSapiens 16 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggagacagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatcagcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtggggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcagcctgcagcct 240 gaagattttg caacttatta ctgtctacag cataatagtt acccgctcactttcggcgga 300 gggaccaagg tggagatcaa ac 322 17 121 PRT Homo Sapiens 17Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 1015 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 2530 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 4045 Gly Tyr Ile Tyr Tyr Thr Trp Thr Ser Asn Tyr Asn Pro Ser Leu Lys 50 5560 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 7075 80 Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 8590 95 Arg Asp Gln Gly Gln Trp Leu Leu Pro Asp Ala Phe Asp Ile Trp Gly100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 18 107 PRT HomoSapiens 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile ArgAsn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys ArgLeu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser LeuGln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn SerTyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10519 364 DNA Homo Sapiens 19 caggtgcagc tgcaggagtc gggcccagga ctggtgaagccttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactactggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattggctat atctattacacttggacctc caactacaac 180 ccctccctca agagtcgcgt caccatatca gtggacacgtccaagaacca gttctccctg 240 aggctgagtt ctgtgaccgc tgcggacacg gccgtttactactgtgcgag agatcagggg 300 cagtggttac tacccgatgc ttttgatatc tggggccaagggacaatggt caccgtctct 360 tcag 364 20 322 DNA Homo Sapiens 20 gacatccagatgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgccgggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagcccctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcggcagtggatc tgggacagag ttcactctca caatcagcag cctgcagcct 240 gaagattttgcaacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300 gggaccaaggtggaaatcaa ac 322 21 123 PRT Homo Sapiens 21 Gln Val Gln Leu Val Gln SerGly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser CysLys Ala Ser Gly Tyr Thr Phe Phe Ser Tyr 20 25 30 Gly Phe Ser Trp Val ArgGln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45 Gly Trp Ile Ser Ala TyrAsn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr MetThr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg SerLeu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr LysVal Arg Gly Val His Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 115 120 22 113 PRT Homo Sapiens 22 Asp IleVal Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 GluArg Ala Thr Ile Ile Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 SerAsn Asn Lys Asn Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 ProPro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 ProAla Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Thr Pro Arg Ser Phe Gly Gln Gly Thr Met Val Glu Ile 100 105110 Lys 23 370 DNA Homo Sapiens 23 caggttcagc tggtgcagtc gggagctgaggtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttttttagctatggtt tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gctgggatggatcagcgctt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatgaccacagaca cttccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgacacggccgtgt attactgtgc gagagaaact 300 aaggttcggg gagtccacta ctacggtatggacgtctggg gccaagggac cacggtcacc 360 gtctcctcag 370 24 340 DNA HomoSapiens 24 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcgagagggccacc 60 atcatctgca agtccagcca gagtatttta tacagctcca acaataagaactacttaggt 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggcatctacccgg 180 gaatccgggg tccctgcccg attcagtggc agcgggtctg ggacagatttcactctcacc 240 atcaacagcc tgcaggctga agatgtggca gtttattact gtcagcaatattatagtact 300 cctcggtcgt tcggccaagg gaccatggtg gaaatcaaac 340 25 119PRT Homo Sapiens 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val LysPro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly SerIle Asn Ser Gly 20 25 30 Gly Cys Tyr Trp Ser Trp Ile Arg Gln His Pro GlyLys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Ser Ser Gly Ser Thr TyrTyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Thr Leu Ser Val Asp Thr SerLys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Asn Ser Met Thr Ala Ala AspThr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg Glu Thr Ala Gly Phe AspTyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 26 107PRT Homo Sapiens 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln AspIle Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro SerArg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile SerGly Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln TyrAsp Thr Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105 27 358 DNA Homo Sapiens 27 caggtgcagc tgcaggagtc gggcccaggactggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcaacagtggtggtt gctactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggattgggtacatct attccagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgaattaccttatcagtag acacgtctaa gaaccagttc 240 tccctgaagc tgaactctat gactgccgcggacacggccg tgtattactg tgcgagagat 300 cgggaaacag ctggttttga ctactggggccagggaaccc tggtcaccgt ctcctcag 358 28 322 DNA Homo Sapiens 28 gacatccagatgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgccaggcgagtca ggacattaac aactatttaa attggtatca gcagaaacca 120 gggaaagcccctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180 aggttcagtggaagtggatc tgggacagat tttactttca ccatcagcgg cctgcagcct 240 gaggatattgcaacatatta ctgtcaacag tatgatactc tccctctcac tttcggcggc 300 gggaccaaggtggagatcaa ac 322 29 120 PRT Homo Sapiens 29 Gln Val Gln Leu Val Glu SerGly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr AspGly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr IleSer Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile PheGly Val Val Ile Asp Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly ThrThr Val Thr Val 115 120 30 107 PRT Homo Sapiens 30 Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp TyrGln Gln Asn Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala SerThr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser GlyThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp ValAla Thr Tyr Tyr Cys Gln Lys Phe Ser Ser Pro Pro Phe 85 90 95 Thr Phe GlyPro Gly Thr Lys Val Asp Ile Ser 100 105 31 367 DNA Homo Sapiens 31caggtgcagc tggtggagtc ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatgcca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagatcgatt 300tttggagtgg ttatcgacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctcag 367 32 322 DNA Homo Sapiens 32 gacatccaga tgacccagtc tccatcctccctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca gggcattagaaattatttag cctggtatca gcagaatcca 120 gggaaagttc ctaagctcct gatctatggtgcatccactt tgcaatcagg ggtcccatct 180 cggttcagtg gcagtggatc tgggacagatttcactctca ccatcagcag cctgcagcct 240 gaagatgttg caacttatta ctgtcaaaagtttagcagtc ccccattcac tttcggccct 300 gggaccaaag tggatatcag tc 322 33 117PRT Homo Sapiens 33 Gln Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val LysPro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly SerIle Ser Ser Gly 20 25 30 Thr Tyr His Trp Ser Trp Ile Arg Gln His Pro GlyArg Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr TyrHis Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr SerLys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala AspThr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Asp Gly Tyr Arg Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 34 107 PRT HomoSapiens 34 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser ProGly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile SerAsn Asn 20 25 30 Phe Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg LeuLeu Ile 35 40 45 Phe Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser LeuGln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn AsnTrp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10535 352 DNA Homo Sapiens 35 caggtgcagc tggagcagtc ggggccagga ctggtgaagccttcagagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtggtacttaccactggag ctggatccgc 120 cagcacccag ggaggggcct ggagtggatt ggatacatctattacagtgg gagcacctac 180 cacaacccgt ccctcaagag tcgaattacc atatcagtagacacgtctaa gaaccagttc 240 tccctgaagc tgagctctgt gacggccgcg gacacggccgtgtattactg tgcgagaggg 300 ggagatggct acagatactg gggccaggga accctggtcaccgtctcctc ag 352 36 322 DNA Homo Sapiens 36 gaaatagtga tgacgcagtctccagccacc ctgtctgtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtcagagtattagc aacaacttcg cctggtacca gcagaaacct 120 ggccaggctc ccaggctcctcatctttggt gcatccacca gggccactgg tatcccagcc 180 aggttcagtg gcagtgggtctgggacagaa ttcactctca ccatcagcag cctacagtct 240 gaagattttg cagtttattactgtcagcag tataataact ggcctcggac gttcggccaa 300 gggaccaagg tggaaatcaa ac322 37 121 PRT Homo Sapiens 37 Gln Val Gln Leu Gln Glu Ser Gly Pro GlyLeu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val SerGly Gly Ser Ile Ser Thr Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro ProGly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Thr Gly Asn ThrTyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Val Ser Val Asp ThrSer Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Asn Ser Val Thr Ala AlaAsp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Pro Gly Gln Trp Leu ValPro Asp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Ser ValSer Ser 115 120 38 112 PRT Homo Sapiens 38 Asp Ile Val Met Thr Gln SerPro Leu Ser Leu Pro Val Ile Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile SerCys Arg Ser Ser Gln Ser Leu Leu Gln Ser 20 25 30 Asn Gly Asn Asn Tyr LeuAsp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile TyrLeu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu AlaAsp Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Ile Pro LeuThr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 39 364 DNA HomoSapiens 39 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cctcggagaccctgtccctc 60 acctgcactg tctctggtgg ctccatcagt acttactact ggagttggatccggcagccc 120 ccagggaagg gactggagtg gattggatac atctattaca ctgggaacacctactacaac 180 ccctccctca agagtcgagt caccgtttca gttgacacgt ccaagaaccagttctccctg 240 aagctgaact ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgagagatccaggc 300 cagtggctgg tccctgatgc ttttgatatc tggggccaag ggacaatggtctccgtctct 360 tcag 364 40 337 DNA Homo Sapiens 40 gatattgtga tgactcagtctccactctcc ctgcccgtca ttcctggaga gccggcctcc 60 atctcctgca ggtctagtcagagcctcctg cagagtaatg gaaacaacta tttggattgg 120 tacctgcaga agccagggcagtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggttcagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgacgatgttgggatt tattactgca tgcaagctct ccaaattcct 300 ctcactttcg gcggagggaccaaggtggag atcaaac 337

What is claimed:
 1. A method of inhibiting cell proliferation associatedwith the expression of MUC18 tumor antigen comprising: providing amonoclonal antibody comprising a heavy chain amino acid, wherein saidantibody has an amino acid sequence selected from the group consistingof SEQ ID NOs: 1,5 9, 13, 17, 21, 25, 29, 33 and 37, and wherein saidmonoclonal antibody binds MUC18; contacting cells expressing MUC18 withan effective amount of said antibody; and incubating said cells and saidantibody, wherein said incubation results in inhibited proliferation ofsaid cells.
 2. The method of claim 1, wherein said antibody is a fullyhuman antibody.
 3. The method of claim 1, wherein said antibody furthercomprises a light chain amino acid having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34 and
 38. 4. The method of claim 1, wherein said antibody isconjugated to a therapeutic or cytotoxic agent.
 5. The method of claim4, wherein the cytotoxic agent is ricin.
 6. The method of claim 4,wherein the further therapeutic agent is a radioisotope.
 7. The methodof claim 1, wherein said cell is a melanoma cell.
 8. The method of claim1, wherein said cell is a tumor cell.
 9. The method of claim 1, whereinsaid cell proliferation is tumor metastasis.
 10. A method of inhibitingcell proliferation associated with the expression of MUC18 tumor antigencomprising: providing a monoclonal antibody comprising a light chainamino acid, wherein said antibody has an amino acid sequence selectedfrom the group consisting of: SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30,34 and 38, and wherein said monoclonal antibody binds MUC18; contactingcells expressing MUC18 with an effective amount of said antibody; andincubating said cells and said antibody, wherein said incubation resultsin inhibited proliferation of said cells.
 11. The method of claim 10,wherein said antibody is a fully human antibody.
 12. The method of claim10, wherein said antibody is conjugated to a therapeutic or cytotoxicagent.
 13. The method of claim 13, wherein the cytotoxic agent is ricin.14. The method of claim 13, wherein the further therapeutic agent is aradioisotope.
 15. The method of claim 10, wherein said cell is amelanoma cell.
 16. The method of claim 10, wherein said cell is a tumorcell.
 17. The method of claim 10, wherein said cell proliferation istumor metastasis.